The cancer suppressor gene, testis-specific protein Y-encoded-like 5 (TSPYL5), has been implicated in various cancers, including colorectal cancer (CRC). In this study, we investigated the role of TSPYL5 in the development of CRC in vitro. First, we used bioinformatics to predict the binding target of TSPYL5, and found that the microRNA, miR-19-5p, bound to the 3' untranslated region (UTR) of TSPYL5. This interaction was further validated by the dual-luciferase assay. Second, we found that overexpressed TSPYL5 enhanced apoptosis in HT29 cells and reduced cell proliferation, reduced cell migration/invasion, and most of the cells accumulated in the G0/G1 phase of the cell cycle. These effects were reversed after addition of miR-19-5p mimics. Third, knocking down expression of miR-19-5p also increased apoptosis, and reduced cell proliferation, migration, and invasion in HT29 cells. We speculate that miR-19-5p induces the degradation of TSPYL5 by binding to its 3'UTR. Our results suggest that increasing the expression of TSPYL5 in HT29 cells or inhibiting miR-19-5p promotes apoptosis of HT29 cells. Thus, miR-19-5p could be used as biomarkers of CRC, with potential implications for diagnosis and therapeutic intervention.